Vanadate substituted phytase: immobilization, structural characterization and performance for sulfoxidations

Area of Science:

• Biocatalysis
• Enzyme engineering
• Organic chemistry

Background:

• 3-phytase is an enzyme with potential applications in biocatalysis.
• Cross-linked enzyme aggregates (CLEAs) offer advantages in enzyme stability and reusability.
• Vanadate is a metal ion that can interact with enzyme active sites.

Purpose of the Study:

• To synthesize and characterize a 3-phytase CLEA for asymmetric sulfoxidation.
• To investigate the role of vanadate in the enzyme's activity and selectivity.
• To assess the impact of organic solvents on the enzyme's conformation and activity.

Main Methods:

• Synthesis of 3-phytase cross-linked enzyme aggregate (CLEA).
• Asymmetric sulfoxidation of thioanisole using hydrogen peroxide as the oxidant.
• (51)V NMR and circular dichroism (CD) spectroscopies to study vanadate incorporation and enzyme conformation.

Main Results:

• The 3-phytase-CLEA exhibited high conversion (ca. 95%) and enantioselectivity (ca. 60%), comparable to the free enzyme.
• The CLEA demonstrated reusability over at least three cycles without significant activity loss.
• Vanadate incorporation altered the enzyme's alpha-helical content, and organic solvents like acetonitrile reduced enzyme activity by decreasing this content.

Conclusions:

• 3-phytase CLEA is an efficient and reusable biocatalyst for asymmetric sulfoxidation.
• Vanadate binding influences the enzyme's active site and conformation, affecting enantioselectivity.
• Organic solvents negatively impact phytase activity by altering enzyme structure, limiting their use in certain applications.